ORIGINAL RESEARCH published: 05 January 2016 doi: 10.3389/fmicb.2015.01486 Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem Jorge A. Valdivia-Anistro1, Luis E. Eguiarte-Fruns1, Gabriela Delgado-Sapién2, Pedro Márquez-Zacarías3, Jaime Gasca-Pineda1, Jennifer Learned4, James J. Elser4, Gabriela Olmedo-Alvarez5 and Valeria Souza1* 1 Laboratorio de Evolución Molecular y Experimental, Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Coyoacán, Mexico, 2 Laboratorio de Genómica Bacteriana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Coyoacán, Mexico, 3 School of Biology, Georgia Institute of Technology, Atlanta, GA, USA, 4 School of Life Sciences, Arizona State University, Tempe, AZ, USA, 5 Laboratorio de Bacteriología Molecular, Departamento de Ingeniería Genética, CINVESTAV – Unidad Irapuato, Irapuato, Mexico The ribosomal RNA (rrn) operon is a key suite of genes related to the production Edited by: of protein synthesis machinery and thus to bacterial growth physiology. Experimental Roland Hatzenpichler, evidence has suggested an intrinsic relationship between the number of copies of this California Institute of Technology, USA operon and environmental resource availability, especially the availability of phosphorus Reviewed by: (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons Sebastian Kopf, Princeton University, USA and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic Albert Leopold Mueller, ecosystem that contains an unusually high microbial diversity that is able to persist Stanford University, USA under highly oligotrophic conditions. These environmental conditions impose a variety *Correspondence: Valeria Souza of strong selective pressures that shape the genome dynamics of their inhabitants. The [email protected] genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6–15 copies). The Specialty section: This article was submitted to main goal of this study was to analyze the variation in the number of rrn operon copies Microbial Physiology and Metabolism, of Bacillus in the CCB and to assess their growth-related properties as well as their a section of the journal Frontiers in Microbiology stoichiometric balance (N and P content). We defined 18 phylogenetic groups within the Received: 16 September 2015 Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The Accepted: 09 December 2015 growth dynamic of these Bacilli was heterogeneous and did not show a direct relation Published: 05 January 2016 to the number of operon copies. Physiologically, our results were not consistent with Citation: the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from Valdivia-Anistro JA, Eguiarte-Fruns LE, growth rate. However, we speculate that the diversity of the growth properties of these Delgado-Sapién G, Bacilli as well as the low P content of their cells in an ample range of rrn copy number Márquez-Zacarías P, Gasca-Pineda J, Learned J, Elser JJ, is an adaptive response to oligotrophy of the CCB and could represent an ecological Olmedo-Alvarez G and Souza V mechanism that allows these taxa to coexist. These findings increase the knowledge (2016) Variability of rRNA Operon of the variability in the number of copies of the rrn operon in the genus Bacillus and Copy Number and Growth Rate Dynamics of Bacillus Isolated from an give insights about the physiology of this bacterial group under extreme oligotrophic Extremely Oligotrophic Aquatic conditions. Ecosystem. Front. Microbiol. 6:1486. doi: 10.3389/fmicb.2015.01486 Keywords: rRNA operon copies, oligotrophy, bacterial growth, Bacillus, Cuatro Ciénegas Frontiers in Microbiology | www.frontiersin.org 1 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics INTRODUCTION copy number may be favored because the rich P supply could then support the rapid production of P-rich rrn to meet the Population genetics is the most direct tool for use in protein demands of rapid growth, as stated by the “Growth understanding adaptation to the ecological challenges imposed Rate Hypothesis” (GRH), a core idea within the theory of upon microbial communities by the environment (Whitaker biological stoichiometry (Elser et al., 2000; Elser, 2006). However, et al., 2003; Xu, 2006; Spencer et al., 2008). Functional traits can in environments with low phosphorus availability, multiple aid in the study of population genetics, because they help to define rrn operon copies could represent a competitive cost if a species in terms of their ecological roles, such as how they use high rrn operon copy number leads to the over-production environmental resources or how they interact with other species of P-rich rrn (Sterner and Elser, 2002; Maciá, 2005; Jeyasingh (McGill et al., 2006; Hughes et al., 2008). These functional traits and Weider, 2007). Indeed, aquatic bacteria isolated from are often considered to be ecological strategies because they are oligotrophic environments usually have low rrn operon copy useful in understanding why certain bacteria live in a particular numbers (Fegatella et al., 1998; Strehl et al., 1999; Lauro et al., environment and how they respond to environmental challenges 2009), as well as various other adaptations to decrease cellular (Green et al., 2008). phosphorus demand (Cavicchioli et al., 2003; Alcaraz et al., The ribosomal RNA operon (rrn hereafter) is the key genetic 2008; Martiny et al., 2009; Van Mooy et al., 2009). Thus, it has structure for protein synthesis and thus a functional trait been proposed that there is a connection between the number related to bacterial life history (Stevenson and Schmidt, 2004). of rrn operon copies and environmental P availability (Elser Ecologically, the rrn operon has been related with the bacterial et al., 2000; Weider et al., 2005; Jeyasingh and Weider, 2007). capacity to respond to changes in environmental conditions However, to our knowledge, we lack extensive studies that (Codon et al., 1995; Prüβ et al., 1999; Green et al., 2008). In document this variation in rrn operon copy number and other particular, the variation in the number of copies of the rrn associated ecological strategies employed by bacteria coexisting operon has been considered an ecological strategy related to in oligotrophic environments, especially those characterized by resource availability, with physiological implications associated severe P limitation. with bacterial growth rate and fitness (Klappenbach et al., 2000; The aims of this study were to describe rrn operon Shrestha et al., 2007). The rrn operon is comprised of three genes copy number variation in different lineages of Bacillus strains (5S, 16S, and 23S rDNA) and its copy number varies from 1 to isolated from an extremely oligotrophic ecosystem and to 15 among bacterial genomes (Klappenbach et al., 2001; Acinas analyze the possible association between the copy number and et al., 2004; Stoddard et al., 2015) and even more dramatically its physiological implications for growth rate and chemical among eukaryotes (Elser et al., 2000). Experimentally, it has been composition (P content and N:P stoichiometry). Severe P shown that deletions of one or more copies of the rrn operon limitation is considered a primary selective pressure that drives have a considerable impact on growth rate, affecting various bacterial evolution in this environment (Souza et al., 2008, 2012). stress-response mechanisms (Nanamiya et al., 2010; Yano et al., For example, we have previously reported on an endemic and 2013). Hence, it has been suggested that the multiplicity of the moderately halophilic Bacillus (type strain of B. coahuilensis: rrn operon is a potential mechanism for adaptation to different m4-4 = NRRL B-41737T), (Cerritos et al., 2008)thathasa environmental conditions (Elser et al., 2000; Green et al., 2008). typical number of rrn copies (nine) but also clear adaptations to In general terms, bacteria that possess more rrn operon copies the extreme oligotrophic conditions, including a small genome may cope better with fluctuating nutrient inputs than bacteria (3.5 Mb), a diversity of phosphate acquisition genes (Moreno- with fewer rrn operon copies, which tend to live in environments Letelier et al., 2011), and a cellular membrane composed of where nutrients are scarce (Klappenbach et al., 2001; Elser, 2003; sulfolipids (Alcaraz et al., 2008). Similar adaptations to low Jeyasingh and Weider, 2007). Moreover, the relationship between P levels have been reported only from oligotrophic marine rrn operon copy number and the bacterial biotic potential for cyanobacteria with low rrn copy numbers (Cavicchioli et al., the cellular allocation of key resources could be analogous to 2003; Lauro et al., 2009; Martiny et al., 2009; Van Mooy et al., the ecological strategies described in other macro-biota (r- and 2009). Hence, the present study represents the first attempt to link K-strategies), (Pianka, 1970; Elser et al., 2000; Dethlefsen and biological stoichiometry to Bacillus diversity and rrn operon copy Schmidt, 2007; Shrestha et al., 2007; Lipowsky et al., 2012). number, as well as the first report in which the numbers of rrn Bacillus is a genus that is well-known because of its copies are analyzed